CHAPTER 2.2.1.
CRAYFISH PLAGUE (APHANOMYCES ASTACI)
1.
Scope
For the purpose of this chapter, crayfish plague is considered to be infection of crayfish with Aphanomyces astaci
Schikora.
2.
Disease information
2.1. Agent factors
2.1.1.
Aetiological agent, agent strains
The aetiological agent of crayfish plague is Aphanomyces astaci. Aphanomyces astaci is a member of
a group of organisms commonly known as the water moulds. Although long regarded to be fungi, this
group, the Oomycetida, are now considered protists and are classified with diatoms and brown algae in
a group called the Stramenopiles or Chromista.
Four groups (A–D) of A. astaci have been described based on random amplification of polymorphic
DNA polymerase chain reaction (RAPD PCR) (Huang et al., 1994; Dieguez-Uribeondo et al. 2005):
Group A (the so called Astacus strains) comprises a number of strains that were isolated from Astacus
astacus and Astacus leptodactylus; these strains are thought to have been in Europe for a long period
of time. Group B (Pacifastacus strains I) includes isolates from both A. astacus in Sweden and
Pacifastacus leniusculus from Lake Tahoe, USA. Imported P. leniusculus have probably introduced
A. astaci and infected the native A. astacus in Europe. Group C (Pacifastacus strains II) consists of a
strain isolated from P. leniusculus from Pitt Lake, Canada. Another strain (Pc), isolated from
Procambarus clarkii in Spain, sits in group D (Procambarus strain). This strain shows
temperature/growth curves with higher optimum temperatures compared with isolates from northern
Europe (Dieguez Uribeondo et al., 1995). Aphanomyces astaci strains that have been present in
Europe for many years (group A strains) appear to be less pathogenic than strains introduced more
recently with crayfish imports from North America since the 1960s.
2.1.2.
Survival outside the host
Although A. astaci is not an obligate parasite and will grow well under laboratory conditions on artificial
media (Alderman & Polglase, 1986; Cerenius et al., 1988), in the natural environment it does not
survive well for long periods in the absence of a suitable host.
Aphanomyces astaci zoospores remain motile for up to 3 days and cysts survive for 2 weeks in distilled
water (Svensson & Unestam, 1975; Unestam, 1966). As A. astaci can go through three cycles of
zoospore emergence, the maximum life span outside of a host could be several weeks. Spores
remained viable in a spore suspension kept at 2°C for 2 months (Unestam, 1966).
2.1.3.
Stability of the agent (effective inactivation methods)
Aphanomyces astaci, both in culture and in infected crayfish, is killed by a short exposure to
temperatures of 60°C or to temperatures of –20°C (or below ) for 48 hours (or more) (Alderman, 2000;
Oidtmann et al., 2002). Sodium hypochlorite and iodophors are effective for disinfection of
contaminated equipment. Equipment must be cleaned prior to disinfection, since organic matter was
found to decrease the effectiveness of iodophors (Alderman & Polglase, 1985). Thorough drying of
equipment (>24 hours) is also effective as A. astaci is not resistant to desiccation.
2.1.4.
Life cycle
The life cycle of A. astaci is simple with vegetative hyphae invading and ramifying through host tissues,
eventually producing extramatrical sporangia that release amoeboid primary spores. These initially
encyst, but then release a biflagellate zoospore (secondary zoospore). Biflagellate zoospores swim in
the water column and, on encountering a susceptible host, attach and germinate to produce invasive
vegetative hyphae. Free-swimming zoospores appear to be chemotactically attracted to crayfish cuticle
(Cerenius & Söderhäll, 1984a) and often settle on the cuticle near a wound (Nyhlen & Unestam, 1980).
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Zoospores are capable of repeated encystment and re-emergence, extending the period of their
infective viability (Cerenius & Söderhäll, 1984b). Growth and sporulation capacity is strain-and
temperature-dependent (Dieguez Uribeondo et al., 1995).
2.2. Host factors
2.2.1.
Susceptible host species
To date, all species of freshwater crayfish have to be considered as susceptible to infection with
A. astaci. The outcome of an infection varies depending on species. All stages of European crayfish
species, including the Noble crayfish (Astacus astacus) of north-west Europe, the white clawed crayfish
(Austropotamobius pallipes) of south-west and west Europe, the related Austropotamobius torrentium
(mountain streams of south-west Europe) and the slender clawed or Turkish crayfish (Astacus
leptodactylus) of eastern Europe and Asia Minor are highly susceptible (Alderman, 1996; Alderman et
al., 1984; Rahe & Soylu, 1989; Unestam, 1969b; 1976; Unestam & Weiss, 1970). Laboratory
challenges have demonstrated that Australian species of crayfish are also highly susceptible
(Unestam, 1976). North American crayfish such as the signal crayfish (Pacifastacus leniusculus),
Louisiana swamp crayfish (Procambarus clarkii) and Orconectes spp. are infected by A. astaci, but
under normal conditions the infection does not cause clinical disease or death. All North American
crayfish species investigated to date have been shown to be susceptible to infection, demonstrated by
the presence of the pathogen in host cuticle (Oidtmann et al., 2006; Unestam, 1969b; Unestam &
Weiss, 1970) and it is therefore currently assumed that this is the case for any other North American
species.
The only other crustacean known to be susceptible to infection by A. astaci is the Chinese mitten crab
(Eriocheir sinensis) but this was reported only under laboratory conditions (Benisch, 1940).
2.2.2.
Susceptible stages of the host
All live stages need to be considered as susceptible to infection.
2.2.3.
Species or subpopulation predilection (probability of detection)
Highly susceptible species: in natural outbreaks of crayfish plague, moribund and dead crayfish of a
range of sizes (and therefore ages) can be found.
North American crayfish species: the prevalence of infection tends to be lower in animals that have
gone through a recent moult (B. Oidtmann, unpublished data). However, large scale systematic studies
have not been undertaken to corroborate these observations. Juvenile crayfish go through several
moults per year, whereas adult crayfish usually moult at least once per year in temperate climates.
Therefore, animals in which the last moult was some time ago may show higher prevalence compared
with animals that have recently moulted.
2.2.4.
Target organs and infected tissue
The tissue that becomes initially infected is the exoskeleton cuticle. Soft cuticle, as is found on the
ventral abdomen and around joints, is preferentially affected. In the highly susceptible European
crayfish species, the pathogen often manages to penetrate the basal lamina located underneath the
epidermis cell layer. From there, A. astaci spreads throughout the body primarily by invading
connective tissue and haemal sinuses; however, all tissues may be affected.
In North American crayfish species, infection is usually restricted to the cuticle. Based on PCR results,
the tailfan (consisting of uropds and telson) and soft abdominal cuticle appear to be frequently infected
(Oidtmann et al., 2006; Vrålstad et al., 2011).
2.2.5.
Persistent infection with lifelong carriers
A number of North American crayfish species have been investigated for their susceptibility to infection
with A. astaci and disease (Oidtmann et al., 2006; Unestam, 1969a; Unestam & Söderhäll, 1977). So
far, infection has been consistently shown in all North American crayfish species tested to date.
Animals investigated were usually clinically healthy.
This is supported by a recent study where the chances of detecting an A. astaci positive signal crayfish
were shown to increase significantly with increasing crayfish length. Furthermore, large female crayfish
expressed significantly higher levels of A. astaci than large males (Vrålstad et al., 2011). The results
probably reflect the decreased moult frequency of larger mature individuals compared with smaller
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immature crayfish (Reynolds, 2002), where mature females tend to moult even less frequently than
mature males (Skurdal & Qvenild, 1986).
Based on the observations made in North American crayfish species, it seems reasonable to assume
that all crayfish species native to the North American continent can be infected with A. astaci without
development of clinical disease and they may therefore act as lifelong carriers of the pathogen.
2.2.6.
Vectors
There is good field and experimental evidence that movements of fish from areas in which crayfish
plague is active can transmit infection from one watershed to another (Alderman et al., 1987; Oidtmann
et al., 2006).
Fomites: A. astaci can also be spread by contaminated equipment (nets, boots, clothing etc.).
2.2.7.
Known or suspected wild aquatic animal carriers
A number of studies have shown that crayfish species native to North America can act as carriers of
A. astaci (e.g. signal crayfish, spiny cheek crayfish, red swamp crayfish (Alderman et al., 1990;
Oidtmann et al., 2006). Since all North American species tested to date have been shown to be
potential carriers of the disease, it is also assumed that North American species not tested to date are
likely to act potentially as carriers of A. astaci. North American species are wide spread in several
regions of Europe. A recent report from Finland also suggests that low density Noble crayfish
populations in cold water environments may be infected at low levels in a chronic infection (ViljamaaDirks et al., 2011).
2.3. Disease pattern
2.3.1.
Transmission mechanisms
The main routes of spread of the pathogen are through 1) movement of infected crayfish, 2) movement
of spores with contaminated water or equipment, as may occur during fish movements, or 3) through
colonisation of habitats by North American crayfish species.
Transmission from crayfish to crayfish occurs, in short, through the release of zoospores from an
infected animal and attachment of such zoospores to a naïve crayfish. The zoospores of A. astaci swim
actively in the water column and have been demonstrated to show positive chemotaxis towards
crayfish (Cerenius & Söderhäll, 1984a).
The main route of spread of crayfish plague in Europe between the 1960s and 2000 was through the
active stocking of North American crayfish into the wild or escapes from crayfish farms (Alderman,
1996; Dehus et al., 1999). Nowadays, spread mainly occurs through expanding populations of North
American crayfish, accidental co-transport of specimens, and release of North American crayfish into
the wild by private individuals (Edsman, 2004; Oidtmann et al., 2005).
Colonisation of habitats, initially occupied by highly susceptible species, by North American crayfish
species carrying A. astaci is likely to result in an epidemic among the highly susceptible animals. The
velocity of spread will depend, among other factors, on the prevalence of infection in the population of
North American crayfish.
Fish transports may facilitate the spread of A. astaci in a number of ways, such as through the
presence of spores in the transport water, A. astaci surviving on fish skin, co-transport of infected
crayfish specimens, or a combination of all three (Alderman et al., 1987; Oidtmann et al., 2002). There
is also circumstantial evidence of spread by contaminated equipment (nets, boots clothing, etc.).
2.3.2.
Prevalence
In the highly susceptible European crayfish species, exposure to A. astaci spores is considered to lead
to infection and eventually to death. The minimal infectious dose has still not been established, but it
may be as low as a single spore per animal (B. Oidtmann, unpublished data). Prevalence of infection
within a population in the early stage of an outbreak may be low (only one or a few animals in a river
population may be affected). However, the pathogen is amplified in affected animals and subsequently
released into the water; usually leading to 100% mortality in a contiguous population. The velocity of
spread from initially affected animals depends on several factors, one being water temperature
(Oidtmann et al., 2005). Therefore, the time from first introduction of the pathogen into a population to
noticeable crayfish mortalities can vary greatly and may range from a few weeks to months.
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Prevalence of infection will gradually increase over this time and usually reach 100%. Data from a
Noble crayfish population in Finland that experienced an outbreak of crayfish plague in 2001 that was
followed in subsequent years suggest that in sparse Noble crayfish populations, spread throughout the
host population may be prolonged over a time span of several years.
Prevalence levels in North American crayfish appear to vary greatly. Limited studies suggest
prevalence levels ranging from anywhere between 0 and 100% are possible (Oidtmann et al., 2006).
2.3.3.
Geographical distribution
First reports of large crayfish mortalities go back to 1860 in Italy (Ninni, 1865; Seligo, 1895). These
were followed by further reports of crayfish mortalities, where no other aquatic species were affected,
in the Franco-German border region in the third quarter of the 19th century. From there a steady
spread of infection occurred, principally in two directions: down the Danube into the Balkans and
towards the Black Sea, and across the North German plain into Russia and from there south to the
Black Sea and north-west to Finland and, in 1907, to Sweden. In the 1960s, the first outbreaks in Spain
were reported, and in the 1980s further extensions of infection to the British Isles, Turkey, Greece and
Norway followed (Alderman, 1996). The reservoir of the original infections in the 19th century was
never established; Orconectes spp. were not known to have been introduced until the 1890s, but the
post-1960s extensions are largely linked to movements of North American crayfish introduced more
recently for purposes of crayfish farming (Alderman, 1996). Escapes of such introduced species were
almost impossible to prevent and Pacifastacus leniusculus and Procambarus clarkii are now widely
naturalised in many parts of Europe.
Since North American crayfish serve as a reservoir of A. astaci, any areas where North American
crayfish species are found have to be considered as areas where A. astaci is present (unless shown
otherwise).
Australia and New Zealand have never experienced any outbreaks of crayfish plague to date and are
currently considered free of the disease (OIE WAHID website, accessed June 2011).
2.3.4.
Mortality and morbidity
When the infection first reaches a naïve population of highly susceptible crayfish species, high levels of
mortality are usually observed within a short space of time, so that in areas with high crayfish densities
the bottoms of lakes, rivers and streams are covered with dead and dying crayfish. A band of mortality
will spread quickly from the initial outbreak site downstream, whereas upstream spread is slower.
Where population densities of susceptible crayfish are low fewer zoospores will be produced, the
spread of infection will be slower and evidence of mortality less dramatic. Water temperature has some
effect on the speed of spread and this is most evident in low-density crayfish populations where
animal-to-animal spread takes longer and challenge intensity will be lower. Lower water temperatures
and reduced numbers of zoospores are associated with slower mortalities and a greater range of
clinical signs in affected animals (Alderman et al., 1987). Observations from Finland suggest that at low
water temperatures, noble crayfish can be infected for several months without the development of
noticeable mortalities (S. Viljamaa-Dirks, unpublished data).
On rare occasions, single specimens of the highly susceptible species have been found after a wave of
crayfish plague has gone through a river or lake. This is most likely to be due to lack of exposure of
these animals during an outbreak (animals may have been present in a tributary of a river/lake or in a
part of the affected river/lake that was not reached by spores, or crayfish may have stayed in burrows
during the crayfish plague wave). However, low-virulent strains of crayfish plague have been described
to persist in a water way, kept alive by a weak infection in the remnant population (Viljamaa-Dirks et al.,
2011). Although remnant populations of susceptible crayfish species remain in many European
watersheds, the dense populations that existed 150 years ago are now heavily diminished (Alderman,
1996; Souty-Grosset et al., 2006). Populations of susceptible crayfish may re-establish, but once
population density and geographical distribution is sufficient for susceptible animals to come into
contact with infection, new outbreaks of crayfish plague in the form of large-scale mortalities will occur.
2.3.5.
Environmental factors
Under laboratory conditions, the preferred temperature range at which the A. astaci mycelium grows
slightly varies depending on the strain. In a study, which compared a number of A. astaci strains that
had been isolated from a variety of crayfish species, mycelial growth was observed between 4 and
29.5°C, with the strain isolated from Procambarus clarkii growing better at higher temperatures
compared to the other strains. Sporulation efficiency was similarly high for all strains tested between
4 and 20°C, but it was clearly reduced for the non-P. clarkii strains at 25°C and absent at 27°C. In
contrast, sporulation still occurred in the P. clarkii strain at 27°C. The proportion of motile zoospores
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(out of all zoospores observed in a zoospore suspension) was almost 100% at temperatures ranging
from 4–18°C, reduced to about 60% at 20°C and about 20% at 25°C in all but the P. clarkii strain. In
the P. clarkii strain, 80% of the zoospores were still motile at 25°C, but no motile spores were found at
27°C (Dieguez Uribeondo et al., 1995).
Field observations show that crayfish plague outbreaks occur at a wide temperature range, and at least
in the temperature range from 4–20°C. The velocity of spread within a population depends on several
factors, including water temperature. In a temperature range between 4 and 16°C, the speed of an
epidemic is enhanced by higher water temperatures. At low water temperatures, the epidemic curve
can increase very slowly and the period during which mortalities are observed can be several months
(B. Oidtmann, unpublished data).
In buffered, redistilled water, sporulation occurs between pH 5 and 8, with the optimal range being
pH 5–7. The optimal pH range for swimming of zoospores appears to be in a pH range from 6.–7.5,
with a maximum range between pH 4.5 and 9.0 (Unestam, 1966).
Zoospore emergence is influenced by the presence of certain salts in the water. CaCl2 stimulates
zoospore emergence from primary cysts, whereas MgCl2 has an inhibitory effect. In general, zoospore
emergence is triggered by transferring the vegetative mycelium into a medium where nutrients are
absent or low in concentration (Cerenius & Söderhäll, 1984b).
2.4. Control and prevention
Once A. astaci has been introduced into a population of highly susceptible crayfish species in the wild, the
spread within the affected population cannot be controlled. Therefore, prevention of introduction is essential.
To avoid the main pathways of introduction, the following measures are necessary:
1.
Movements of potentially infected live or dead crayfish, potentially contaminated water, equipment or
any other item that might carry the pathogen from an infected to an uninfected site holding susceptible
species should be prevented.
2.
When fish transfers are being planned, it should be considered whether the source water may harbour
infected crayfish (including North American carrier crayfish).
3.
Any fish movements from the site of a current epidemic of crayfish plague carries a high risk of spread
and should generally be avoided.
4.
If fish movements from a source containing North American crayfish are being planned, fish harvest
methods at the source site need to ensure that: a) crayfish are not accidentally co-transported; b) the
transport water does not carry A. astaci spores, and, c) equipment is disinfected between use; d) the
consignment does not become contaminated during transport.
5.
The release of North American crayfish into the wild in areas where any of the highly susceptible
species are present should be prevented. Once released, North American crayfish tend to spread,
sometimes over long distances. Therefore prior to any planned release, careful consideration needs to
be given to the long-term potential consequences of such a release. Highly susceptible crayfish
populations at a distance from the release site may eventually be affected.
6.
Aquaculture facilities for the cultivation of crayfish are very rarely suitable for preventing the spread of
crayfish from such sites. Therefore, careful consideration needs to be given, as to whether such
facilities should be established.
Certain pathways of introduction, such as the release of North American crayfish by private individuals are
difficult to control.
2.4.1.
Vaccination
Currently, there is no evidence that vaccines offer long-term protection in crustaceans and even if this
were not to be the case, vaccination of natural populations of crayfish is impossible.
2.4.2.
Chemotherapy
No treatments are currently known that can successfully treat the highly susceptible crayfish species,
once infected.
2.4.3.
Immunostimulation
No immunostimulants are currently known that can successfully protect the highly susceptible crayfish
species against infection and consequent disease due to A. astaci infection.
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2.4.4.
Resistance breeding
In the 125 years since crayfish plague first occurred in Europe, there is little evidence of resistant
populations of European crayfish. However, the fact that North American crayfish are not very
susceptible to developing clinical disease suggests that selection for resistance may be possible and
laboratory studies using A. astaci strains attenuated for virulence might be successful. However, there
are currently no published data referring to such studies.
2.4.5.
Restocking with resistant species
North American crayfish have been used in various European countries to replace the lost stocks of
native crayfish. However, since North American crayfish are potential hosts for A. astaci, restocking
with North American crayfish would further the spread of A. astaci. Given the high reproduction rates
and the tendency of several North American crayfish species to colonise new habitats, restocking with
North American crayfish species would largely prevent the re-establishment of the native crayfish
species.
2.4.6.
Blocking agents
No data available.
2.4.7.
Disinfection of eggs and larvae
Limited information is available on the susceptibility of crayfish eggs to infection with A. astaci.
Unestam & Söderhäll mention that they experimentally exposed Astacus astacus and P. leniusculus
eggs to zoospore suspensions and were unable to induce infection (Unestam & Söderhäll, 1977).
However, the details of these studies have not been published.
Although published data are lacking, disinfection of larvae, once infected, is unlikely to be successful,
since A. astaci would be protected from disinfection by the crayfish cuticle, in which it would be
present.
2.4.8.
General husbandry practices
If a crayfish farm for highly susceptible species is being planned, it should be carefully investigated
whether North American crayfish species are in the vicinity of the planned site or whether North
American crayfish populations may be present upstream (for sites that are “online” on a stream or
abstracting water from a stream), even if at a great distance upstream. If North American crayfish are
present, there is a high likelihood that susceptible farmed crayfish will eventually become infected.
In an established site, where the highly susceptible species are being farmed, the following
recommendations should be followed to avoid an introduction of A. astaci onto the site:
3.
1.
Movements of potentially infected live or dead crayfish, potentially contaminated water,
equipment or any other item that might carry the pathogen from an infected to an uninfected
site holding susceptible species must be prevented.
2.
If fish transfers are being planned, these must not come from streams or other waters that
harbour potentially infected crayfish (either susceptible crayfish populations that are going
through a current outbreak of crayfish plague or North American carrier crayfish).
3.
North American crayfish must not be brought onto the site.
4.
Fish obtained from unknown freshwater sources or from sources, where North American
crayfish may be present or a current outbreak of crayfish plague may be taking place, must
not be used as bait or feed for crayfish, unless they have been subject to a temperature
treatment that will kill A. astaci (see Section 2.1.3).
5.
Any equipment that is brought onto site should be disinfected.
6.
General biosecurity measures should be in place (e.g. controlled access to premises;
disinfection of boots when site is entered; investigation of mortalities if they occur; introduction
of live animals (crayfish, fish) only from sources known to be free of crayfish plague).
Sampling
3.1. Selection of individual specimens
In the case of a suspected outbreak of crayfish plague in a population of highly susceptible crayfish species,
the batch of crayfish selected for investigation for the presence of A. astaci should ideally consist of: a) live
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crayfish showing signs of disease, b) live crayfish appearing to be still healthy, and, c) dead crayfish that
may also be suitable, although this will depend on their condition.
Live crayfish should be transported using polystyrene containers equipped with small holes to allow aeration,
or an equivalent container. The temperature in the container should not exceed 16°C.
The container should provide insulation against major temperature differences outside the container. In
periods of hot weather, freezer packs should be used to avoid temperatures deleterious to the animals.
These can be attached at the inside bottom of the transport container. The crayfish must however be
protected from direct contact with freezer packs. This can be achieved using, for instance, cardboard or a
several layers of newspaper.
Crayfish should be transported in a moist atmosphere, for example using moistened wood shavings/wood
wool, newspaper or grass/hay. Unless transport water is sufficiently oxygenated, live crayfish should not be
transported in water, as they may suffocate from lack of oxygen.
The time between sampling of live animals and delivery to the investigating laboratory should not exceed
24 hours.
Should only dead animals be found at the site of a suspected outbreak, these might still be suitable for
diagnosis. Depending on the condition they are in, they can either be: a) transported chilled (if they appear
to have died only very recently), or, b) placed in non-methylated ethanol (minimum concentration 70%; see
Section 3.2).
Animals showing advanced decay are unlikely to give a reliable result, however, if no other animals are
available, these might still be tested.
3.2. Preservation of samples for submission
The use of non-preserved crayfish is preferred, as described above. If, for practical reasons, transport of
recently dead or moribund crayfish cannot be arranged quickly, crayfish may be fixed in ethanol (minimum
70%). However, fixation may reduce test sensitivity. The crayfish:ethanol ratio should ideally be 1:10 (1 part
crayfish, 10 parts ethanol).
3.3. Pooling of samples
Not recommended.
3.4. Best organs or tissues
In highly susceptible species, the tissue recommended for sampling is the soft abdominal cuticle, which can
be found on the ventral side of the abdomen.
In the North American crayfish species, sampling of soft abdominal cuticle, uropods and telson are
recommended.
3.5. Samples/tissues that are not suitable
Autolytic material is not suitable for analysis.
4.
Diagnostic methods
Large numbers of dead crayfish of the highly susceptible species with the remaining aquatic fauna being
unharmed gives rise to a suspicion that the population may be affected by crayfish plague. Clinical signs of
crayfish plague include behavioural changes and a range of visible external lesions. However, clinical signs are of
limited diagnostic value. The main available diagnostic methods are polymerase chain reaction (PCR) and
isolation of the pathogen in culture media followed by confirmation of its identity. Isolation can be difficult and
requires that samples are in good condition when they arrive at the investigating laboratory (Oidtmann et al.,
1999). Molecular methods are now available that are less dependent on speed of delivery and can deal with a
greater range of samples compared with methods relying on agent isolation (Oidtmann et al., 2006; Vrålstad et
al., 2009).
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Chapter 2.2.1. — Crayfish plague (Aphanomyces astaci)
4.1. Field diagnostic methods
4.1.1.
Clinical signs
Highly susceptible species
Gross clinical signs are extremely variable and depend on challenge severity and water temperatures.
The first sign of a crayfish plague mortality may be the presence of numbers of crayfish at large during
daylight (crayfish are normally nocturnal), some of which may show evident loss of co-ordination in
their movements, and easily fall over on their backs and remain unable to right themselves. Often,
however, unless waters are carefully observed, the first sign that there is a problem will be the
presence of large numbers of dead crayfish in a river or lake (Alderman et al., 1987)
In susceptible species, where sufficient numbers of crayfish are present to allow infection to spread
rapidly, particularly at summer water temperatures, infection will spread quickly and stretches of over
50 km may lose all their crayfish in less than 21 days from the first observed mortality (D. Alderman,
pers. comm.). Crayfish plague has unparalleled severity of effect, since infected susceptible crayfish
generally do not survive. It must be emphasised, however, that the presence of large numbers of dead
crayfish, even in crayfish plague-affected watersheds, is not on its own sufficient for diagnosis. The
general condition of other aquatic fauna must be assessed. Mortality or disappearance of other aquatic
invertebrates, as well as crayfish, even though fish survive, may indicate pollution (e.g. insecticides
such as cypermethrin have been associated with initial misdiagnoses).
North American crayfish species
Melanised cuticle has sometimes been suggested as a sign of infection with A. astaci. However,
melanisation can have a wide variety of causes and is not a specific sign of A. astaci infection.
Conversely, animals without signs of melanisation are often infected.
4.1.2.
Behavioural changes
Highly susceptible species
Infected crayfish of the highly susceptible crayfish species may leave their hides during daytime (which
is not normally seen in crayfish), have a reduced escape reflex, and progressive paralysis. Dying
crayfish are sometimes found lying on their backs. The animals are often no longer able to upright
themselves. Occasionally, the infected animals can be seen trying to scratch or pinch themselves.
North American crayfish species
Infected North American crayfish do not show any behavioural changes (B. Oidtmann, unpublished
data).
4.2. Clinical methods
4.2.1.
Gross pathology
Highly susceptible species
Depending on a range of factors, the foci of infection in crayfish may be seen by the naked eye or may
not be discernable despite careful examination. Such foci can best be seen under a low power stereo
microscope and are most commonly recognisable by localised whitening of the muscle beneath the
cuticle. In some cases a brown colouration of cuticle and muscle may occur, and in others, hyphae are
visible in infected cuticles in the form of fine brown (melanised) tracks in the cuticle itself. Sites for
particular examination include the intersternal soft ventral cuticle of the abdomen and tail, the cuticle of
the perianal region, the cuticle between the carapace and abdomen, the joints of the pereiopods
(walking legs), particularly the proximal joint and finally the gills.
North American crayfish species
Infected North American crayfish can sometimes show melanised spots in their soft cuticle, for
example the soft abdominal cuticle. However, it must be stressed that these melanisations can be
caused by mechanical injuries or infections with other water moulds and are very unspecific.
Conversely, visible melanisation is not always associated with carrier status. Infected animals can
appear completely devoid of visible melanisations.
4.2.2.
Clinical chemistry
No suitable methods available.
108
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Chapter 2.2.1. — Crayfish plague (Aphanomyces astaci)
4.2.3. Microscopic pathology
Unless the selection of tissue for fixation has been well chosen, A. astaci hyphae can be difficult to find
in stained preparations. Additionally, such material does not prove that any hyphae observed are those
of the primary pathogen. A histological staining technique, such as the Grocott silver stain
counterstained with conventional haematoxylin and eosin, can be used.
See also Section 4.2.4.
4.2.4. Wet mounts
Small pieces of soft cuticle excised from the regions mentioned above (Section 4.2.1) and examined
under a compound microscope using low to medium power will confirm the presence of aseptate fungal
hyphae 7–9 µm wide. The hyphae can usually be found pervading the whole thickness of the cuticle,
forming a three-dimensional network of hyphae in heavily affected areas of the cuticle. The presence of
host haemocytes and possibly some melanisation closely associated with and encapsulating the
hyphae give good presumptive evidence that the hyphae represent a pathogen rather than a
secondary opportunist invader. In some cases, examination of the surface of such mounted cuticles will
demonstrate the presence of characteristic A. astaci sporangia with clusters of encysted primary
spores (see Section 4.3).
4.2.5. Smears
Not suitable.
4.2.6. Fixed sections
See section 4.2.3.
4.2.7. Electron microscopy/cytopathology
Not suitable.
4.3. Agent detection and identification methods
4.3.1.
Direct detection methods
4.3.1.1. Microscopic methods
4.3.1.1.1. Wet mounts
As indicated above (Section 4.2.4.), presumptive identification of A. astaci may be made from the
presence of hyphae pervading the cuticle and sporangia of the correct morphological types (see
below) on the surface of crayfish exoskeletons.
4.3.1.1.2. Smears
Not suitable.
4.3.1.1.3. Fixed sections
See Section 4.2.3.
4.3.1.2. Agent isolation and identification
4.3.1.2.1. Cell culture/artificial media
Highly susceptible species
Care should be taken that animals to be used for isolation of A. astaci via cultivation are not
exposed to desiccation.
Isolation methods have been described by Benisch (1940); Nyhlen & Unestam (1980); Alderman &
Polglase (1986); Cerenius et al. (1988); Oidtmann et al. (1999) and Viljamaa-Dirks (2006).
Isolation medium (IM) according to Alderman & Polglase (1986): 12.0 g agar; 1.0 g yeast extract;
5.0 g glucose; 10 mg oxolinic acid; 1000 ml river water; and 1.0 g penicillin G (sterile) added after
autoclaving and cooling to 40°C. River water is defined as any natural river or lake water, as
opposed to demineralised water.
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Chapter 2.2.1. — Crayfish plague (Aphanomyces astaci)
Any superficial contamination should first be removed from the soft intersternal abdominal cuticle or
any other areas from which cuticle will be excised by thoroughly wiping the cuticle with a wet (using
autoclaved H2O) clean disposable paper towel. Simple aseptic excision of infected tissues, which
are then placed as small pieces (3–5 mm2) on the surface of isolation medium plates , will normally
result in successful isolation of A. astaci from moribund or recently dead (<24 hours) animals.
Depending on a range of factors, foci of infection in crayfish may be easily seen by the naked eye
or may not be discernable despite careful examination. Such foci can best be seen under a lowpower stereo microscope and are most commonly recognisable by localised whitening of the
muscle beneath the cuticle. In some cases, a brown colouration of cuticle and muscle may occur,
and in others, hyphae are visible in infected cuticles in the form of fine brown (melanised) tracks in
the cuticle itself. Sites for particular examination include the intersternal soft ventral cuticle of the
abdomen and tail, the cuticle of the perianal region, the cuticle between the carapace and tail, the
joints of the pereiopods (walking legs), particularly the proximal joint and finally the gills.
Provided that care is taken in excising infected tissues for isolation, contaminants need not present
significant problems. Small pieces of cuticle and muscle may be transferred to a Petri dish of sterile
water and there further cut into small pieces with sterile instruments for transfer to isolation medium
(IM). Suitable instruments for such work are scalpels, fine forceps and scissors.
To reduce potential contamination problems, disinfection of the cuticle with ethanol and melting a
sterile glass ring 1–2 mm deep into the isolation medium can improve isolation success (Nyhlen &
Unestam, 1980; Oidtmann et al., 1999). The addition of potassium tellurite into the area inside the
glass ring has been described (Nyhlen & Unestam, 1980).
Inoculated agar can be incubated at temperatures between 16°C and 24°C. The Petri dishes
should be sealed with a sealing film (e.g. Parafilm1) to avoid desiccation.
On IM agar, growth of new isolates of A. astaci is almost entirely within the agar except at
temperatures below 7°C, when some superficial growth occurs. Colonies are colourless.
Dimensions and appearance of hyphae are much the same in crayfish tissue and in agar culture.
Vegetative hyphae are aseptate and (5)7–9(10) µm in width (i.e. normal range 7–9 µm, but
observations have ranged between 5 and 10 µm). Young, actively growing hyphae are densely
packed with coarsely granular cytoplasm with numerous highly refractile globules (Alderman &
Polglase, 1986). Older hyphae are largely vacuolate with the cytoplasm largely restricted to the
periphery, leaving only thin strands of protoplasm bridging the large central vacuole. The oldest
hyphae are apparently devoid of contents. Hyphae branch profusely, with vegetative branches often
tending to be somewhat narrower than the main hyphae for the first 20–30 µm of growth.
When actively growing thalli or portions of thalli from broth or agar culture are transferred to river
water (natural water with available cations encourages sporulation better than distilled water),
sporangia form readily in 20–30 hours at 16°C and 12–15 hours at 20°C. Thalli transferred from
broth culture may be washed with sterile river water in a sterile stainless steel sieve, before transfer
into fresh sterile river water for induction of sporulation. Thalli in agar should be transferred by
cutting out a thin surface sliver of agar containing the fungus so that a minimum amount of nutrientcontaining agar is transferred. Always use a large volume of sterile river water relative to the
amount of fungus being transferred (100:1). Sporangia are myceloid, terminal or intercalary,
developing from undifferentiated vegetative hyphae. The sporangial form is variable: terminal
sporangia are simple, developing from new extramatrical hyphae, while intercalary sporangia can
be quite complex in form. Intercalary sporangia develop by the growth of a new lateral extramatrical
branch, which forms the discharge tube of the sporangium. The cytoplasm of such developing
discharge tubes is noticeably dense, and these branches are slightly wider (10–12 µm) than
ordinary vegetative hyphae. Sporangia are delimited by a single basal septum in the case of
terminal sporangia and by septa at either end of the sporangial segment in intercalary sporangia.
Such septa are markedly thicker than the hyphal wall and have a high refractive index. Successive
sections of vegetative hypha may develop into sporangia, and most of the vegetative thallus is
capable of developing into sporangia.
Within developing sporangia, the cytoplasm cleaves into a series of elongate units (10–25 × 8 µm)
that are initially linked by strands of protoplasm. Although the ends of these cytoplasmic units
become rounded, they remain elongate until and during discharge. Spore discharge is achlyoid,
that is, the first spore stage is an aplanospore that encysts at the sporangial orifice and probably
represents the suppressed saprolegniaceous primary zoospore. No evidence has been found for
1
110
Reference to specific commercial products as examples does not imply their endorsement by the OIE. This applies to all
commercial products referred to in this Aquatic Manual.
Manual of Diagnostic Tests for Aquatic Animals 2012
Chapter 2.2.1. — Crayfish plague (Aphanomyces astaci)
the existence of a flagellated primary spore, thus, in this description, the terms ‘sporangium’ not
‘zoosporangium’ and ‘primary spore’ not ‘primary zoospore’ have been used. Discharge is fairly
rapid (<5 minutes) and the individual primary spores (=cytoplasmic units) pass through the tip of the
sporangium and accumulate around the sporangial orifice. The speed of cytoplasmic cleavage and
discharge is temperature dependent. At release, each primary spore retains its elongate irregularly
amoeboid shape briefly before encystment occurs.
Encystment is marked by a gradual rounding up followed by the development of a cyst wall, which
is evidenced by a change in the refractive index of the cell. The duration from release to
encystment is 2–5 minutes. Some spores may drift away from the spore mass at the sporangial tip
and encyst separately. Formation of the primary cyst wall is rapid, and once encystment has taken
place the spores remain together as a coherent group and adhere well to the sporangial tip so that
marked physical disturbance is required to break up the spore mass.
Encysted primary spores are spherical, (8)9–11(15) µm in diameter, and are relatively few in
number, (8)15–30(40) per sporangium in comparison with other Aphanomyces spp. Spores remain
encysted for 8–12 hours. Optimum temperatures for sporangial formation and discharge for the
majority of European isolates of A. astaci are between 16 and 24°C (Alderman & Polglase, 1986).
For some isolates, particularly from Spanish waters, slightly higher optimal temperatures may
prevail (Dehus et al., 1999). The discharge of secondary zoospores from the primary cysts peaks at
20°C and does not occur at 24°C. In new isolates of A. astaci, it is normal for the majority of primary
spore cysts to discharge as secondary zoospores, although this varies with staling in long-term
laboratory culture. Sporangial formation and discharge occurs down to 4°C. A. astaci does not
survive at –5°C and below for more than 24 hours in culture, although –20°C for >48 hours may be
required in infected crayfish tissues, nor does it remain viable in crayfish tissues that have been
subject to normal cooking procedures (Alderman, 2000; Oidtmann et al., 2002).
In many cases, some of the primary spores are not discharged from the sporangium and many
sporangia do not discharge at all. Instead, the primary spores appear to encyst in situ within the
sporangium, often develop a spherical rather than elongate form and certainly undergo the same
changes in refractive index that mark the encystment of spores outside the sporangium. This withinsporangial encystment has been observed on crayfish. Spores encysted in this situation appear to
be capable of germinating to produce further hyphal growth.
Release of secondary zoospores is papillate, the papilla developing shortly before discharge. The
spore cytoplasm emerges slowly in an amoeboid fashion through a narrow pore at the tip of a
papilla, rounds up and begins a gentle rocking motion as a flagellar extrusion begins and the spore
shape changes gradually from spherical to reniform. Flagellar attachment is lateral (Scott, 1961);
zoospores are typical saprolegniaceous secondary zoospores measuring 8 × 12 µm. Active motility
takes some 5–20 minutes to develop (dependent on temperature) and, at first, zoospores are slow
and uncoordinated. At temperatures between 16 and 20°C, zoospores may continue to swim for at
least 48 hours (Alderman & Polglase, 1986).
Test sensitivity and specificity of the cultivation method can be very variable depending on the
experience of the examiner, but in general will be lower than the PCR.
North American crayfish species
Isolation of A. astaci by culture following the methods described for the highly susceptible species
usually fails. Currently, the recommended method for detecting infection in such species is by PCR.
4.3.1.2.2. Antibody-based antigen detection methods
None available.
4.3.1.2.3. Molecular techniques
Animals
In the case of a suspected outbreak of the disease in highly susceptible crayfish species, moribund
or recently dead (<24 hours) crayfish are preferably selected for DNA extraction. Live crayfish can
be killed using chloroform. If the only animals available are animals that have died a few days prior
to DNA extraction, they can be tested, but a negative PCR result must be interpreted with caution
as DNA degradation may have occurred. Endogenous controls can be used to assess whether
degradation may have occurred. These should preferably use host tissues richer in host cells
compared to the cuticle; cuticle itself contains very few host cell nuclei. If circumstances prevent
delivery of crayfish to the specialist laboratory within 24 hours, fixation in 70% ethanol (≥3:1 ethanol
to crayfish tissue) is possible, but may result in a reduction of the DNA yield.
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Chapter 2.2.1. — Crayfish plague (Aphanomyces astaci)
DNA extraction
Where animals of the highly susceptible species are analysed, the soft abdominal cuticle is the
preferred sample tissue for DNA extraction. Any superficial contamination should first be removed
by thoroughly wiping the soft abdominal cuticle with a wet (using autoclaved H2O) clean disposable
paper towel. The soft abdominal cuticle is then excised and 30–50 mg ground in liquid nitrogen to a
fine powder using a pestle and mortar (alternative grinding techniques may be used, but should be
compared with the liquid nitrogen method before routine use). For carrier identification, 30–50 mg
tissue from each soft abdominal cuticle, and telson and uropods are sampled and processed
separately. DNA is extracted from the ground cuticle using a proteinase K-based DNA extraction
method (e.g. DNeasy tissue kit; Qiagen, Hilden, Germany; protocol for insect tissue) following the
manufacturer's instructions (Oidtmann et al., 2006) or using a CTAB (cetyltrimethylammonium
bromide-based)-based assay (Vrålstad et al., 2009). Negative controls should be run alongside the
samples. Shrimp tissues may be used as negative controls.
4.3.1.2.3.1. PCR
Several PCR assays have been developed with varying levels of sensitivity and specificity. Two
assays are described here that have proven highly sensitive and specific. Both assays target the
ITS (internal transcribed spacer) region of the nuclear ribosomal gene cluster within the A. astaci
genome. As should be standard for any PCR-based diagnostic tests, negative controls should be
run alongside the samples to control for potential contamination. Environmental controls (using for
example shrimp tissue as described above) and extraction blank controls from the DNA extraction
should be included along with ‘no template’ PCR controls (template DNA replaced with molecular
grade water). The no template PCR controls should include an environmental PCR control left open
during pipetting of sample DNA.
Method 1:
This conventional PCR assay uses species-specific primer sites located in the ITS1 and ITS2
regions. Forward primer (BO 42) 5’-GCT-TGT-GCT-GAG-GAT-GTT-CF-3’ and reverse primer (BO
640) 5’-CTA-TCC-GAC-TCC-GCA-TTC-TG-3’. The PCR is carried out in a 50 µl reaction volume
containing 1 × PCR buffer 75 mM Tris/HCl, pH 8.8, 20 mM [NH4]2SO4, 0.01% (v/v) Tween 20),
1.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dTTP, and dGTP, 0.5 µM of each primer, and
1.25 units of DNA polymerase (e.g. Thermoprime Plus DNA Polymerase; AB Gene, Epsom, UK) or
equivalent Taq polymerase and 2 µl DNA template. The mixture is denatured at 96°C for 5 minutes,
followed by 40 amplification cycles of: 1 minute at 96°C, 1 minute at 59°C and 1 minute at 72°C
followed by a final extension step of 7 minutes at 72°C. Amplified DNA is analysed by agarose gel
electrophoresis. The target product is a 569 bp fragment. Confirmation of the identity of the PCR
product by sequencing is recommended. The assay consistently detects down to 500 fg of genomic
target DNA or the equivalent amount of ten zoospores submitted to the PCR reaction (Tuffs &
Oidtmann, 2011).
Method 2:
This assay is a TaqMan minor groove binder (MGB) real-time PCR assay that targets a 59 bp
unique sequence motif of A. astaci in the ITS1 region. Forward primer AphAstITS-39F (5’-AAGGCT-TGT-GCT-GGG-ATG-TT-3’), reverse primer AphAstITS-97R (5’-CTT-CTT-GCG-AAA-CCTTCT-GCT-A-3’) and TaqMan MGB probe AphAstITS-60T (5’-6-FAM-TTC-GGG-ACG-ACC-CMGBNF-Q-3’) labelled with the fluorescent reporter dye FAM at the 5’-end and a non-fluorescent
quencher MGBNFQ at the 3’-end. Real-time PCR amplifications are performed in a total volume of
25 µl containing 12.5 µl PCR Master Mix (e.g. Universal PCR Master Mix or Environmental PCR
Master Mix, Applied Biosystems), 0.5 µM of the forward (AphAstITS-39F) and reverse (AphAstITS97R) primers, 0.2 µM 200 nM of the MGB probe (AphAstITS-60T), 1.5 µl molecular grade water
and 5 µl template DNA (undiluted and tenfold diluted). Amplification and detection is performed in
Optical Reaction Plates sealed with optical adhesive film or similar on a real-time thermal cycler.
The PCR programme consists of an initial decontamination step of 2 minutes at 50°C to allow
optimal UNG enzymatic activity, followed by 10 minutes at 95°C to activate the DNA polymerase,
deactivate the UNG and denature the template DNA, and successively 50 cycles of 15 seconds at
95°C and 60 seconds at 58°C. A dilution series with reference DNA of known DNA content needs
to be run alongside with the samples.
The absolute limit of detection of this assay was reported as approximately 5 PCR forming units (=
target template copies), which is equivalent to less than one A. astaci genome (Vrålstad et al.,
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Chapter 2.2.1. — Crayfish plague (Aphanomyces astaci)
2009). Another study reported consistent detection down to 50 fg using this assay (Tuffs &
Oidtmann, 2011).
The diagnostic test sensitivity of either assay largely depends on the quality of the samples taken.
Where a crayfish plague outbreak is investigated, the test sensitivity in animals that had died of
crayfish plague 12 hours or less prior to sampling or in live crayfish showing clear clinical signs of
disease is expected to be high. Studies to investigate the effect of sensitivity loss caused by
deteriorating sample quality (for instance because of delayed sampling, processing or unsuitable
storage of samples) have not been carried out. It is recommended that multiple (5–10) crayfish be
tested, to compensate for variations in sample quality and invasion site of the pathogen.
Analytical test specificity has been investigated (Oidtmann et al., 2006; Tuffs & Oidtmann, 2011;
Vrålstad et al., 2009) and no cross reaction was observed. However, owing to the repeated
discovery of new Aphanomyces strains, sequencing is recommended to confirm diagnosis. In the
case of the real-time PCR assay, this requires separate amplification of a PCR product, either using
the primers as described in method 1, or using primers ITS 1 and ITS4 (see section ‘sequencing’
below).
4.3.1.2.3.2. Sequencing
A PCR product of 569 bp can be amplified using primers BO42 and BO640. The size of the PCR
amplicon is verified by agarose gel electrophoresis, and purified by excision from this gel (e.g. using
the Freeze n’ Squeeze DNA purification system, Anachem, Luton, UK). Both DNA strands must be
sequenced using the primers used in the initial amplification. The consensus sequence is
generated using sequence analysis software and compared with published sequences using an
alignment search tool such as BLAST. If 100% identify between the submitted sequence and the
published sequences is found, then the amplified product is A. astaci. If the sequence is not 100%
identical, further sequencing should be performed using primers ITS-1 (5’-TCC-GTA-GGT-GAACCT-GCG-G-3’) and ITS-4 (5’-TCC-TCC-GCT-TAT-TGA-TAT-GC-3’) (White et al., 1990), which
generate an amplicon of 757 bp that provides sequence data in the same region, but expanded at
both ends relative to the sequence generated by primers BO42 and BO640. This expanded
sequence should confirm the identity of the pathogen to the species level.
Highly susceptible species
PCR (conventional or real-time) is a suitable method to investigate suspected crayfish plague
outbreaks (see Section 7.1). Where the conditions of a suspect case are fulfilled, amplification of a
PCR product of the expected size using conventional PCR or real-time PCR can be considered
sufficient as a confirmatory diagnosis, if a high level of template DNA is detected. Where low levels
of template DNA are detected (weak amplification) or the samples are investigated from a site not
meeting the conditions of a suspect case, it is recommended to sequence PCR products generated
as described under the section sequencing to confirm the diagnosis.
4.3.1.2.4. Agent purification
None available.
4.3.2.
Serological methods
None available.
5.
Rating of tests against purpose of use
The methods currently available for diagnosis of clinical diseases of crayfish plague (Aphanomyces astaci) in
highly susceptible species are listed in Table 5.1. Methods for targeted surveillance to demonstrate freedom from
disease in highly susceptible species are displayed in Table 5.2.
Clinical disease is extremely rare in North American crayfish. Therefore a rating of methods for diagnosing clinical
disease in these species is not provided. However, methods for targeted surveillance to demonstrate freedom
from infection in North American crayfish are listed in Table 5.3.
The designations used in the tables indicate: a = the method is the recommended method for reasons of
availability, utility, and diagnostic specificity and sensitivity; b = the method is a standard method with good
diagnostic sensitivity and specificity; c = the method has application in some situations, but cost, accuracy, or
other factors severely limits its application; and d = the method is presently not recommended for this purpose.
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Chapter 2.2.1. — Crayfish plague (Aphanomyces astaci)
These are somewhat subjective as suitability involves issues of reliability, sensitivity, specificity and utility.
Although not all of the tests listed as category a or b have undergone formal standardisation and validation, their
routine nature and the fact that they have been used widely without dubious results, makes them acceptable.
Table 5.1. Crayfish plague diagnostic methods in highly susceptible crayfish species
Method
Presumptive diagnosis
Confirmatory diagnosis
Gross and microscopic signs
b
d
Isolation and culture
b
d
Histopathology
d
d
PCR
a
b or a1
qPCR
a
b or a1
Sequencing of PCR products
n/a
a
Transmission EM
d
d
Antibody-based assays
n/a
n/a
DNA probes — in situ
n/a
n/a
PCR = polymerase chain reaction; qPCR = quantitative PCR; EM = electron microscopy; n/a = not applicable or not available;
1 = see definitions of confirmed case in Section 7.1.
Table 5.2. Methods for targeted surveillance in highly susceptible crayfish species to declare freedom from crayfish plague
Method
Screening method
Confirmatory method
Inspection for gross signs and mortality
a
c
Microscopic signs (wet mounts)
c
c
Isolation and culture
c
b
Histopathology
d
d
PCR
a
b, possibly a1
qPCR
a
b, possibly a1
Sequencing of PCR products
n/a
a
Transmission EM
d
d
Antibody-based assays
n/a
n/a
DNA probes — in situ
n/a
n/a
PCR = polymerase chain reaction; qPCR = quantitative PCR; EM = electron microscopy; n/a = not applicable or not available;
1 = see definitions of confirmed case in Section 7.1.
Table 5.3. Methods for targeted surveillance in North American crayfish species to declare freedom from crayfish plague
114
Method
Screening method
Confirmatory method
Gross and microscopic signs
d
d
Isolation and culture
c
c
Histopathology
d
d
PCR
a
b
qPCR
a
b
Sequencing of PCR products
n/a
a
Transmission EM
d
d
Manual of Diagnostic Tests for Aquatic Animals 2012
Chapter 2.2.1. — Crayfish plague (Aphanomyces astaci)
Table 5.3. cont. Methods for targeted surveillance in North American crayfish species
to declare freedom from crayfish plague
Method
Screening method
Confirmatory method
Antibody-based assays
n/a
n/a
DNA probes — in situ
n/a
n/a
PCR = polymerase chain reaction; qPCR = quantitative PCR; EM = electron microscopy; n/a = not applicable or not available
6.
Test(s) recommended for targeted surveillance to declare freedom from crayfish plague
(Aphanomyces astaci)
6.1. Highly susceptible species
Crayfish farms keeping susceptible crayfish would need to be inspected at a frequency outlined in Chapter
2.2.0. A history of no mortalities (this does not include losses due to predation) occurring within the
population over a period of at least 12 months combined with absence of clinical signs, as well as gross and
microscopic pathology at the time of inspection are suitable methods for this purpose. Surveillance of wild
crayfish stocks presents greater problems, especially where the species concerned is endangered. As
movements of fish stocks from infected waters present a risk of disease transmission, monitoring the status
of crayfish populations to confirm that they remain healthy, is necessary.
In a farm setting, an infection with crayfish plague should be noticed relatively quickly, due to a relatively
quick onset of mortalities in the farmed crayfish population.
To undertake targeted surveillance, regular inspections are recommended, where samples are collected if
there is any suspicion of mortality or disease. If moribund or dead animals are found, it is recommended that
samples are analysed by PCR and if PCR returns a positive result, that PCR products generated using
primers 42 and 640, or ITS-1 and -4 are sequenced and the sequences analysed.
6.2. North American crayfish species
In North American crayfish species, animals would need to be sampled and analysed using one of the PCR
assays described above. For reasons of higher sensitivity, the real-time PCR assay is the preferred method.
This applies to both farmed and naturalised stocks, and surveillance programmes need to take into account
the risks of indirect transmission by movements of fish.
7.
Corroborative diagnostic criteria
7.1. Definition of suspect case
Highly susceptible crayfish species
Any extensive mortality solely of the highly susceptible species of freshwater crayfish where all other
aspects of the flora and fauna, particularly other aquatic crustaceans, are normal and healthy.
North American crayfish species
Any population of North American crayfish is generally to be regarded as potentially infected with A. astaci.
7.2. Definition of confirmed case
Highly susceptible crayfish species
Confirmation of presence of A. astaci by PCR or qPCR and sequencing.
Where (1) a crayfish mortality meets the definition of a suspect case and (2) PCR results indicate the
presence of high levels of template DNA (in case of real-time PCR, this corresponds to Ct values ≤ 30), and
(3) the investigated suspect case is not the first case of detection of A. astaci in the country or region, the
PCR result alone may be considered sufficient as a confirmation.
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Chapter 2.2.1. — Crayfish plague (Aphanomyces astaci)
North American crayfish species
Confirmation of presence of A. astaci by PCR or qPCR and sequencing
8.
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*
* *
NB: There are OIE Reference Laboratories for Crayfish plague (Aphanomyces astaci)
(see Table at the end of this Aquatic Manual or consult the OIE web site for the most up-to-date list:
http://www.oie.int/en/our-scientific-expertise/reference-laboratories/list-of-laboratories/ ).
Please contact the OIE Reference Laboratories for any further information on
Crayfish plague (Aphanomyces astaci)
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